Journal of Natural Produrts v01.5 3 , N O .1,pp. 81-86,Jan-Feb 1990
81
BULLATACIN, BULLATACINONE, AND SQUAMONE, A NEW BIOACTIVE ACETOGENIN, FROM THE BARK OF ANNONA SQUAMOSA X.-H. Lr, Y.-H. HUI,J.K. RUPPRECHT,Y.-M. LIU, K.V.WOOD,D . L . SMITH, C.-J. CHANG,and J.L. MCLAUGHLIN+
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Phatmaral Scimces, Purdue Uniwrsity, West Lfayette, lndiana 47907 A s s T ~ ~ . - A c t i v i t y d i r e c t e d fractionation of the stem bark of Annona squamosa, monitoring with brine shrimp lethality, led to the isolation of the highly bioactive acetogenins bullatacin 111 and bullatacinone 121,thus demonstrating a new abundant plant source for these potent compounds. A new keto-monotetrahydrofran acetogenin with a ketolactone terminus, as first seen in bullatacinone 121,was also isolated, characterized by spectral analyses, and named squamone 131.The cytotoxicities of 3 were increased significantly by reduction of the two keto groups to hydroxyls, and the tetrahydrosquamone and bullatacinone 121 both showed selective cytotoxicities to MCF-7 human breast carcinoma. Liriodenine and (-)-kaur- 16-en-19-oic acid were also isolated.
Annona squamosa L. (Annonaceae)is the most widely grown of the tropicallsubtropical Annona fruit trees; it is commonly called sugar apple and is an item of considerable commerce. A number of insecticidal and medicinal uses, including antitumor uses, employing various parts of the plant, have been tabulated (1). A patent has been granted for a chemically uncharacterized insecticide, named annonin, isolated from the seeds (2). Annonin has an identical mol wt (622) to asimicin and its diastereomers, bullatacin [l],and the rolliniastatins ( 3 4 , but is spectrally unidentical to these known acetogenins. However, annonin may be identical with squamosin, a stereochemically undefined 15,24,28-trihydroxy-bis-tetrahydrofuranacetogenin recently described from the seeds (7). During our screening for antitumor plants, extracts of the bark of A . squamosa exerted a high degree of lethality to nauplii in the brine shrimp test (BST)(8)and in our panel of three human tumor cell lines. The EtOH extract (FOO1) of the bark was partitioned to give F005,as previously described (4),in which the bioactivity was concentrated. Successive Si gel columns, monitoring with the BST, led to the known highly bioactive acetogenins bullatacin [l]and bullatacinone [2] (4), thus demonstrating a new, more readily available plant source for these potent compounds. In addition, a novel, but less active, acetogenin, which we have named squamone {3], was isolated and characterized. The known compounds liriodenine and (-)-kau- 16-en- 19-oic acid were also found as in A . bullata (4). Squamone [31 was obtained as a waxy colorless solid with mp 89". The hrcims gave an [MH]+ ion at m/z 595.452 1, consistent with a molecular formula ofC3,H620,. The presence of hydroxyl moieties was obvious by the loss of H,O in the cims and a broad absorption in the ir at 3517 cm-'. The presence of only two hydroxyls was further suggested by two resonances (6 74.04 for C-20 and 73.92 for C- 15) in the region for hydroxyl-bearing carbons in the 13C-nmrspectrum, by a multiplet for two protons (H- 15 and H-20) at 6 3.33 in the 'H-nmr spectrum, and by the formation of a diacetate and a di-trimethylsilyl (TMSi) derivative. The 13C-nmrresonances at 6 82.65 (C- 16) and 82.60 (C- 19)and their correspondlV' , . siting scholar from the Tianjin Institute of Pharmaceutical Industry, 133 Dong Ma Road,Tianjin, People's Republic of China.
Journal of Natural Products
82
I
trans
Wol. 53, No. 1
thieo
n
ing 'H nmr resonances at 6 3.62 (2H) were directly analogous to similar signals in annonacin (9) and goniothalamicin (lo), indicating the presence of a single tetrahydrohran moiety. The placement of the two hydroxyls alpha to the tetrahydrofuran ring was established by 'H-'H COSY experiments that linked the two proton signals at 6 3.62 (for H-15 and H-20) to two proton signals at 3.33 (for H-16 and H-19). The subsequent downfield shift of the signal at 6 3.33 to two multiplets of one proton each at 4.84 and 4.87 in the 'H nmr of the acetate confirmed this assignment. Certain aspects of the 'H-nmr spectra of squamone 131 were quite similar to the spectra of bullatacinone 123 (4). A characteristic signal at 6 1.54 (s, 3H, H-35) suggested a terminal methyl ketone as was first observed in the acetogenins with bullatacinone 121. 'H-nmr (C$,) signals at 6 2.69 (dddd, l H , H-2, J=3.47, 9.35, 9.35, 9.35), 2.51 (dd, l H , H-33a, J = 3 . 4 7 , 18.41), 1.912 (dd, l H , H-33b, J = 9.35, 18.4 l),and 4.00 (dddd, l H , H-4, J = 3.38,4.48, 8.24, 8.24) specifically corresponded to fragment C of bullatacinone 123 (4). This was further substantiated by a 2D 'H-'H COSY experiment (C6D6)that showed connectivities between H-NH-33a and H-33b, H-4/H-5, H-2IH-33a and H-33b, and H-2/H-35a and H-35b, indicative of a y-lactone. These assignments were confirmed in the I3C nmr by resonances at 6 205.4 (C-34), 178.00 (C-l), 78.56 (C-4), 44.21 (C-33), 35.36 (C-2), 25.01 (C-35), and 33.50 (C-3). In addition, the eims of 3 was dominated by the acetyl ion at m/z 43 that originated from the cleavage of the bond between C-33 and C-34. The carbonyl peak for C-34 at 6 205.44 was apparent in the '3C-nmr spectra of 3, trans
threo
threo
3 4 5 6
R=H R=Ac R=TMSi R=TMSi-d,
Jan-Feb 19901
Li et al. : Bioactive Acetogenin
83
but another carbonyl peak (6 2 10.8 1, C-9) was also evident. Two triplets at 6 2.07 ( H l o ) and 1.94 (H-8) in the 'H n m r (C6H6)were indicative of methylene protons adjacent to a keto group located somewhere along the hydrocarbon chain. The positions of the tetrahydrofuran ring and the keto group along the chain were determined by careful analysis of the cims and eims fragments of 3 and its acetate derivative 4 , TMSi derivative 5, and its deutero-TMSi derivative 6 . Also, reduction of the two carbonyls of 3 produced a diastereomeric mixture of derivatives with hydroxyls at C-9 and C-34, and this reduced product (tetrahydrosquamone) 7 afforded a TMSi derivative 8, which was
4 trans
gH3(CH2)11
o,
Q J.
OR
OR
threo
34
y
3
OR
threo
7 R=H 8 R=TMSi
helpful in placing the internal keto group at C-9. Exact mass measurements by hrms of selected key fragments substantiated their identifications. [Tetrahydrosquamone was homogeneous on tlc (three systems) but presumably consists of a mixture of diastereomers due to non-stereoselective reaction of the two carbonyl groups. There was not sufficient material available for a more detailed investigation of this point.] The eims of the TMSi derivative 5 (mol wt 738) produced intense ions at m/z 271.2460 (calcd 271.2457), 467.2826 (calcd 467.2829), 341.2873 (calcd 341.2876), and 397.2409 (calcd 397.2410) which served to locate the presence of the two hydroxyl groups at C-15 and C-20 relative to the tetrahydrofuran ring (Figure 1). The TMSi spectrum also showed an intense ion at m/z 54 1 indicative of alpha cleavage (adjacent to a carbonyl) between C-8 and C-9, but the exact mass for this peak suggested that it originated from TMSi derivatization of the enol at C-9 and cleavage between C- 19 and C-20. The hrcims (isobutane) of tetrahydrosquamone { T f gave an [MH]+ at m/z 599.4873 (calcd 599.4887) for C,,HaO,. Eims of the TMSi derivative of 7 produced the expected ions at mlz 271, 615.3931 (calcd 615.3933), 341, and 545.35 13 (calcd 545.35 14),positioning the tetrahydrofuran ring and determining the location of the reduced carbonyl at C-9 by the presence of ions at mlz 27 1.1725 (calcd 271.1730), 615, and 373.2229 (calcd 373.2230) (Figure 2).
mlz 34 1.
FIGURE 1.
(9)
mlz j P 7 + (9)
-
Eims fragmentation of the TMSi (5) and TMSi-d, (6) derivatives of squamone 131; numbers in parentheses indicate the shift in mass units seen with the TMSi-d, derivative; the elemental compositions of fragments marked with an asterisk (+) were confirmed through exact mass measurements, and all agree within 3 mmu with the calculated values.
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615*
mlz 27 I*-
mlz 615-
mlz 27 1*
0
FIGURE2 .
Eims fragmentations of the TMSi derivative 8 of tetrahydrosquamone the elemental compositions of the fragments marked with an asterisk (*)were confirmed through exact mass measurements, and all agree within 3 mmu with the calculated values.
m;
The relative stereochemistry of the tetrahydrofuran ring and the two adjacent hydroxyls at C-15 and C-20 was deduced by close examination of the nmr data. In the spectra of squamone 131, the I3C-nmr chemical shifts of C-15 and C-20 at 6 7 3 . 9 2 and 7 4 . 0 4 clearly showed a threo relationship between C- 15/C-16 and a threo relationship between C-29/C-20 (1 1). These assignments are based on the '3C-nmr chemical shifts of a pair of model monotetrahydrofuran compounds with adjacent hydroxyl groups of the threo and erythro configuration (11):In squamone diacetate E41, the 'H-nmr chemical shifts of H- 16 and H- 19 at 6 3.975 indicated the trans relationship for H- 16/H- 19 ( 1 2 ) . The trans relationship for H- 16/H- 19 is in agreement with the same assignment in bullatacin [l}( 4 ) and is based on the methods of Hoye and Suhadolnik ( 1 2 ) . Thus, the relative configuration of threo, trans, threo from C - 15 to C-20 is evident. However, the stereochemistry of the chiral centers at C-2 and C-4 remains unsolved; squamone {3}does not exhibit significant absorption in the cd spectrum. From the above data we concluded that the structure of squamone is as illustrated for 3 with the absolute and certain relative stereochemistries remaining undefined. Biological activities of the isolated bullatacin [l],bullatacinone [2], and squamone {3) and the prepared tetrahydrosquamone [TI (stereochemistry undefined) are presented in Table 1. It is noteworthy that bullatacinone {2] showed strong selective activity against the MCF-7 breast carcinoma and that tetrahydrosquamone [TI showed one hundred times the activity of squamone in the MCF-7 cell line. The presence of hydroxyls in place of the keto groups may enhance the bioactivity of the keto-acetogenins. TABLE1.
Bioactivities of Bullatacin [l],Bullatacinone 121, Squamone (31, and Tetrahydrosquamone [7l(ED,, pg/ml).
Bullatacin . . . . . . . Bullatacinone . . . . . . Squamone . . . . . . . Tetrahydrosquamone . . Adriamycin . . . . . . .
. . . . .
. . . . .
.I
BST
. 0.002 . 0.003 2.1
. 2.7 . not tested
'Brine shrimp lethality test. bHuman lung carcinoma. 'Human breast carcinoma. dHuman colon adenocarcinoma.
A-549h 8.99 X 1.44 x 1.34 1.41 X lo-'
6.96 X
MCF-7'
> 10
